Method for preparing a surface with a controlled coverage of nanograde particles

ABSTRACT

The present invention regards nano surfaces and particularly a gradient based nano surface. According to embodiments of the invention a surface bound gradient is created by distributed nanoparticles along a plane surface. This procedure greatly reduces the number of prepared surfaces needed, as well as the methodological error of analysis of adsorption and adhesion phenomena.

FIELD OF THE INVENTION

This invention pertains in general to the field of surface chemistry.More particularly the invention relates to surfaces with nano scaleproperties.

BACKGROUND

The problem with solid surface interactions with living biologicaltissue is a repeating subject within areas of medical technology, e.g.biomaterials, biosensors, and controlled drug delivery. Otherapplication areas are food processing technology and biotechnicalprocess chemistry, areas where wanted or unwanted interactions withbiologically produced substances exist. Thus, there is a constant needfor new materials with improved functions and characteristics, and thereis an increasing need of experimental surface modifications suitable forthe specific application areas.

The field of nanotechnology has made great progresses during the lastdecades, mainly due to the fact that nanostructured materials, with astructure size of 1-1000 nm, have very interesting properties withregards to optimized interaction with biological fluids and livingtissue. Central to this patent application is a recently published paperdescribing a method of making nanostructured solid surfaces withnanostructures around 10 nm [1]. The method includes that flat goldsurfaces are allowed to react with thiol terminated, lineary alkanes(dithiols), binding to the gold surface with one thiol group, whereasthe other thiol end will constitute an overlayering carpet of pristinethiol groups.

A stable colloidal solution of negatively charged gold particles in thesize range of 8-12 nm was brought into contact with the surface and thegold particles were adsorbed to the aforementioned pristine thiolgroups. It was observed that the distance between the adsorbed particlescould be controlled by varying the ionic strength of the citrate bufferthat was used during the adsorption. The distance (center to center)could be varied between 10-100 nm when the molarity (concentration) ofthe buffer was varied between 10-0 nM as judged from visualizationexperiments with scanning electron microscopy (SEM). Similar resultshave earlier been demonstrated in particle adsorption experiments withelectrostatically stabilized solutions of surface charged polymerparticles to mineral surfaces such as glass, silicon dioxide or mica[2-5]. In addition, similar results have also been presented concerningadsorption of negatively charged gold surface nanoparticles fromelectrostatic stabilized solutions to glass surfaces or silicon dioxidsurfaces that are positively charged due to chemical modifications [6].

The principle of electrostatically controlled particle adsorption isshown in FIG. 2. An electrostatically stabilized solution 202 containingsurface charged nanoparticles 200 is applied to a beaker 201 (FIG. 2A).A surface preparation 203 is introduced in the container (FIG. 2B)allowing the particles 200 to bind to the surface 203 by means ofelectrostatic, semi-covalent, covalent or other types of bindings,leading to that a stable adsorption is obtained after a certain time(FIG. 2C). The particles have a certain distance, r, from each other.

This condition represents a terminal condition for the adsorption, andprolonged incubation time does not have any further impact on surfacecoverage of particles. When the surface is removed from the particlesolution, the distance between two adjacent particles can be estimatedfrom the interaction pair-potential according to the DLVO-theory [1,7],FIG. 3.

In short, the pair potential U(r), where r is the distance between twoparticles, may be calculated as the sum of an attractive potentialU_(attraction)(r) emanating from the dispersive forces between theparticles as well as a repulsive potential U_(repulsion)(r) emanatingfrom the electrostatic repulsion between the particles.

The shape of the repulsive potential can be calculated in differentways, but will always vary with the so called Debye-distance which is anapproximate measure of the declination of the potential outside thesurface of the particle. A short Debye-distance means that the repulsivepotential is rapidly declining outside the surface of the particle. TheDebye distance is in turn dependent of the ionic strength in theparticle solution 202, and can be expressed as:

$\kappa^{- 1} = \left\lbrack \frac{{ɛɛ}_{0}{kT}}{1000\; e^{2}N_{A}2\; I} \right\rbrack^{1/2}$

where ε is the relative permittivity, ε₀ is the permittivity I invacuum, k is the Boltzmann constant, T is the temperature, e is theelementary charge, N_(A) is Avogadro's constant and the ionic strengthis:

$I = {\frac{1}{2}{\sum\limits_{i = 1}^{n}\; {c_{i}z_{i}^{2}}}}$

where c_(i) and z_(i) is the molar concentration and the charge of an iion in the solution.

The Debye-distance, and the range of the repulsive potential, istherefore reduced with increasing ionic concentration in the colloidalsolution 202. This means that each particle can bind closer to otherparticles on the surface when the condition

$\frac{U(r)}{kT} = \frac{1}{\lambda}$

where U(r) is the pair potential, kT is the thermal energy, and λ is aconstant, is fulfilled for smaller r.

Surfaces prepared with dithiols and gold nanoparticles as describedabove and in [1, 8] have been used in biological experiments. In theseexperiments, the spaces between the particles were made proteinrepelling with a conjugated maleimide reagent which rapidly bindscovalently to dithiol groups. The maleimides were conjugated withpolyethylene glycol (PEG) which resulted in the spaces between theparticles becoming repelling for proteins and cells. The surface on theabsorbed gold particles could later be modified with thiol reagents,e.g. thiol with methyl groups, which gives the adsorbed gold particleshydrophobic characteristics. Surfaces with gold particles made in thisway has a very highly controlled chemical structure and physicalorganization in the nanometer range which makes such surfaces wellsuited for adhesion studies of different kinds. The described method isvery flexible for adhesion studies due to the relatively large number ofcommercial substances with malemide functions which can bind between theadsorbed particles, as well as thiol reagents which can bind to theadsorbed gold particles.

Similar experiments have been performed where gold nanoparticles,stabilized by polymers, have been applied to silica and glass surfacesby so called “dip-coat” technology [9]. Note that this method does notutilize electrostatic repulsion between particles to control theirspread throughout the surface but instead the distance is defined by thepolymer structures that surround the particles in solution. Interactionbetween these particles and the adsorbed surface is weak, why theparticles after adsorption must be sintered in the substrate, a processin which also the surrounding polymers disappears from the surface. Thesurface around the gold particles then becomes the underlying silicasubstrate which can be modified with functional silanes, e.g.PEG-modified silanes, which makes this surface resistant to bioadhesion.The particles' surfaces can be modified with thiol reagents, e.g. thiolconjugated to so called RGD-peptides, an amino acid sequence whichmediates cell interactions.

In this experiment it has also been described that polymer particles inan electrostatic stabilized solution is adsorbed to charged mineralsurfaces and that the distance between the adsorbed particles has beencontrolled with electrostatic repulsion according to the abovedescription [10]. The adsorbed surfaces either have a native net charge,or have been charged through chemical modification, e.g. with functionalsilanes. The bond between the surface and particles has primarily beenof electrostatic nature. The surfaces with the adsorbed polymerparticles have been used as a lithographic template with which thepolymer particle covered parts of the surface have been transformed toisles of gold in the size range of 10-1000 nm surrounded by thesubstrate surface material. The surrounding substrate surface was thenmodified, e.g. with poly-L-lysine-PEG. Lysine is a positively chargedpolymer, which is adsorbed by negatively charged surfaces, and whenconjugated with PEG, in certain cases make these surfaces resistant tobio adhesion. The gold surfaces can then be modified with thiolreagents, e.g. linear alkane thiols, which make the gold surfaceshydrophobic. To these hydrophobic surfaces proteins can be adsorbed,e.g. the protein laminin. Such surfaces have been used to study cellproliferation and surface interaction.

All of the above described technologies can be used to study theimportance of a surface nanostructure for the adhesion process, and canbe used as a platform for the design of materials with desiredbiological characteristics.

Most adhesion studies are performed on surfaces with a constant chemicalsetup. When studying the importance of one type of surface modificationit is common practice to use several surface preparations in order toanalyze adhesion phenomena independently. This procedure, however, istime and labor consuming since several surface preparations must beprepared for each series of experiments. In addition, the methodologicalerrors of measurements can be relatively large which means that theinterpretation of the intended study of adhesion phenomena can be eitherincorrect or overlooked.

A method to limit the methodological error and to reduce time spent toprepare surfaces is to create gradients in the chemical characteristicson a surface. One example of such a method is the so called “wettabilitygradient”, a surface which is hydrophobic in one end and hydrophilic inthe other [11]. Between these endpoints the controlled and continuousgradient of chemical characteristics is found. This type of surfacegradient significantly reduces time to prepare as well as themethodological error, and is often used in academic research [12-14].

Several methods to prepare continuous chemical gradients on a surfaceare known, one of them the well known diffusion method, FIG. 1. In thiscase the method of action is such that a reagent 001, e.g.methylchlorosilane is mixed in a solvent with high density 002, e.g.trichloroethyleneacetate (tri-). The mixture is then layered under adifferent solvent 003, e.g. xylene with low density. Between theselayers there is a surface 004, e.g. glass on which a gradient will form.In time the solvents start to diffuse into each other where also the setof reagent 001 diffuses and binds to the surface 004. At a specific timeof diffusion a bound gradient of hydrophobic methyl groups has occurredon the hydrophilic glass surface [11]. How much of the reagent thatbinds to the surface at a certain position, and therefore thehydrophobicity at this position is determined by the concentration ofthe set of reagents 001 on the surface at this position in combinationwith the time during which the surface has been exposed to the reagentsolution. This means that the characteristics of the obtained gradientare determined through kinetic control.

To manufacture an even gradient in particle density with the abovedescribed method should be difficult, since the binding of nanoparticlesfrom an electrostatic stabilized solution to a surface which binds theseparticles usually is a very fast process in relation to the particlesrate of diffusion. This is low compared to the rate of diffusion forsmall molecules, such as methylchlorosilane. Attempts made to controlthe distance between nanoparticles on surfaces, where nanoparticles hasbeen adsorbed to binding surfaces from electrostatic stabilizedsolutions, through varying the particle concentration and time ofincubation, has shown the difficulties in controlling low densitygradients of particles. Also, the particles do not show the sameconformity of organization on the surface as they do after electrostaticcontrolled adsorption as described above [6, 15].

Recently, a gradient of gold particles on a silica substrate wasdisclosed, where the structuring of bound particles was good [16]. Thisgradient, described in [16] was manufactured according to a modified“dip-coat”-method, but without using electrostatic control or diffusiongradients. The obtained gradient had limited dynamic and the smallestparticle distance was about 50 nm. The gradients were modifiedchemically with PEG between the particles and RGD peptides on top of theparticles. The gradient surfaces were subsequently used in experimentsto investigate cellular adhesion. In general, this publication disclosesthat the interest to make surface bound density gradients of goldparticles is great, for reasons mentioned above. The technical solutionto produce gradients according to [16] is however significantly morecomplex than the present invention.

SUMMARY

Accordingly, the present invention preferably seeks to mitigate,alleviate or eliminate one or more of the above-identified deficienciesin the art and disadvantages singly or in any combination and solves atleast the above mentioned problems by providing a method, a surface, aproduct and a use according to the appended claims.

According to a first aspect a method for preparing a continuous gradientof deposited and electrically charged nanoparticles along a solidsurface is provided, wherein the number of deposited and electricallycharged nanoparticles per unit area of the surface is relatively high onone end of the surface and relatively low on the opposite end of thesurface. The distance between the deposited particles, at the time ofdeposition, is regulated through electrostatic repulsion between thenanoparticles in a solution. The degree of electrostatic repulsion ofparticles in the solution is obtained by a diffusion of a salt solutioninto the solution comprising nanoparticles.

This is advantageous, because it allows forming an improved gradient ofnanoparticles on a surface.

The diffusion of salt solution may be obtained by forming a layer of asalt solution with relatively high density and concentration under alayer of substantially salt free solution comprising nanoparticles, andthat the continuous gradient may be regulated by the time of diffusionand concentration of salt in the salt solution.

In an embodiment, the diffusion of the salt solution may be obtained bykeeping the salt solution in a reservoir, placed into contact with thesuspension of nanoparticles further comprising a matrix allowingdiffusion of nanoparticles but prohibiting convection currents, andwhich suspension of nanoparticles is in contact with the solid surface.

This is advantageous, since it allows forming gradients in twodimensions.

The nanoparticles may consist of metal, ceramics, such as glass, orpolymer material.

The solid surface may consist of metal, ceramics, such as glass, orpolymer material.

The bonding forces between the nanoparticles and the surface maycomprises covalent bonds, Coulombic interactions, metal bonds, van derWaals bonds, hydrogen bonds, dipole-dipole bonds or ion-dipole bonds.

In an embodiment, the surface is gold, with bound dithiol reagent andthe nanoparticles are covalently bound to thiol groups of the dithiolmolecules bound to the gold surface.

In an embodiment, negatively charged but non-surface binding particlesare mixed with surface binding nanoparticles.

This is advantageous, since it may improve dispersion and preventcluster formation of the surface binding nanoparticles.

The method may further comprising adding a first separate surface and asecond separate surface to the surface, wherein the first separatesurface has a surface chemistry similar to the nanoparticle and thesecond separate surface has a surface chemistry similar to the surface.

In an embodiment, scale marks are added to the surface.

An advantage with this is that it may simplify microscopic analysis ofadhesion analysis.

According to a second aspect, a surface is provided, with a continuousgradient of deposited and electrically charged nanoparticles.

The gradient length may be between 1 mm and 50 mm.

The nanoparticles may have an average diameter between 10 and 60 nm.

The average distance of the nanoparticles may be about 10-60 nm in oneend of the gradient and about 100-150 nm in the other end of thegradient.

In an embodiment, the gradient is linear.

The nanoparticles and/or the surface may consist of metal, ceramics,such as glass, or polymer material.

The nanoparticles and/or the surface may have a compound conjugated tothem. The compound may be selected from the group consisting of thiolgroups, such as methyl terminated, amino terminated, acid terminated,peptide terminated, saccharide-conjugated or PEG-conjugated thiol, orthiol silane; PEG, such as poly-L-lysine-PEG, PEG-modified silanes,malemide-PEG; and aminosilane.

According to a third aspect, a device for analysis of adhesion phenomenais provided, comprising a gradient surface according to the secondaspect, a first separate surface and a second separate surface, whereinthe surfaces are separated and the first separate surface has a surfacechemistry similar to the nanoparticle and the second separate surfacehas a surface chemistry similar to the surface.

The nanoparticles, the surface, the first separate surface or the secondseparate surface may have the same or different compound/s conjugated tothem, and the compound/s may be selected from the group consisting ofthiol groups, such as methyl terminated, amino terminated, acidterminated, peptide terminated, saccharide-conjugated or PEG-conjugatedthiol, or thiol silane; PEG, such as poly-L-lysine-PEG, PEG-modifiedsilanes, malemide-PEG; and aminosilane.

According to a fourth aspect, use the surface according to the secondaspect, or the device according to third aspect, for adhesion analysisis provided.

The analysis may be based on surface plasmon resonance (SPR),electrochemistry, light microscopy or scanning electron microscopy(SEM).

The present invention provides the advantage over the prior art that itallows forming an improved gradient of nanoparticles on a surface.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects, features and advantages of which the inventionis capable, will be apparent and elucidated from the followingdescription of embodiments of the present invention, reference beingmade to the accompanying drawings, in which

FIGS. 1 and 2 are illustrations of prior art methods;

FIG. 3 is an illustration of the physics of the DLVO theory;

FIG. 4 is an illustration of the method according to an embodiment;

FIG. 5 is an illustration of the method according to another embodiment;

FIGS. 6 to 10 are illustrations of embodiments;

FIG. 11 is an overview of surfaces according to embodiments of theinvention, analyzed with SEM;

FIG. 12 shows results of two gradients, analyzed with SPR;

FIG. 13 is a line scan of a gradient according to an embodiment,together with a line scan for a positive control surface; and

FIGS. 14 to 16 are results of analyses according to embodiments of theinvention.

DESCRIPTION OF EMOBIDMENTS

Several embodiments of the present invention will be described in moredetail below with reference to the accompanying drawings in order forthose skilled in the art to be able to carry out the invention. Theinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. The embodiments do not limit the invention, but theinvention is only limited by the appended patent claims. Furthermore,the terminology used in the detailed description of the particularembodiments illustrated in the accompanying drawings is not intended tobe limiting of the invention.

According to one aspect of the invention, a method for the convenientmanufacture of surfaces with adsorbed nanoparticles with a gradient isprovided. In an embodiment, the method may be described as follows.

1. One-Dimensional Diffusion

In an embodiment according to FIG. 4, a plane surface 203 with theability to bind electrostatic surface charged nanoparticles 200 from anelectrostatically stabilized particle solution is put in a vial 401. Asalt free, or almost salt free, solution 402 with surface chargedparticles 200 is then added to the vial. A salt solution 403 with arelatively high density is carefully layered under the salt freesolution 402 in a way so that the gravity dependent phase level betweenthe solutions 403 and 402 is leveled with the lower part of the surface203. In time, the salt solution 403 will diffuse into the salt freesolution 402 and form a gradient of ionic strength in this.

As will be appreciated by a person skilled in the art, the surface doesnot need to be plane, but may have any kind of curvature or shape.

The electrostatic dependent repulsion between the particles is reducedwhen the ionic strength in the buffer close to the surface 203increases. The particles therefore adsorb gradually closer to each otheron the surface with the highest density of particles closest to theoriginal phase level between the solutions 402 and 403. The lowestdensity of particles is found in the upper layer of the vial where theionic strength is low and therefore the electrostatic repulsion ishighest. After a controlled time of diffusion the solution is emptiedfrom the vial, from below through the same tube 404 which was used whenlayering the salt solution 403 under particle containing salt freesolution 402.

In absence of convection and that the distance from the surface lowerlevel, x=0 mm, to the bottom of the vial is sufficient, and that thedistance from the surface lower level, x=0 mm, to the surface of thesalt free solution 402 is sufficient, and that the diffusion is notallowed to continue for too long, the gradual distribution of the saltconcentration in the solution above the surface 203 can be describedwith Fick's 2nd law of diffusion in one dimension:

$\frac{\partial c}{\partial t} = {D\frac{\partial^{2}}{\partial x^{2}}c}$

wherein, for the above mentioned conditions:

${c\left( {x,t} \right)} = {\frac{1}{2}{c_{0}\left( {1 - {{erf}\frac{x}{2\sqrt{Dt}}}} \right)}}$

where c is the molar salt concentration, x is the position in the vialin the relation x=0 (which coincides with the lower level of the surface203), t is the time of diffusion, c₀ is the salt concentration in thesolution 403 at t=0, and D is the diffusion constant for the salt inquestion.

This means that the length and the slope of the acquired particlegradient can be varied through changing the initial salt concentrationc₀ and the time of diffusion t which gives the method of manufacturinggreat flexibility.

Further development of the invention described in FIG. 5 uses vials 501which allow several surfaces to be used at the same procedure ofdiffusion as described in FIG. 4. An advantage of this method is thatall surfaces in the vial will be exposed to the same solution of goldparticles, molarity of the salt solution, and the time of diffusion.This assures that surfaces can be prepared with a high rate ofconformity at the same preparation.

The method of diffusion described in relation to FIG. 1 and earlierpublications [11] has technical similarities with the describedinvention, but differs on several crucial aspects. The most importantdifferences are: the component, nanoparticles, which will bind to thesurface, is present as a constant concentration and does not diffuse asa gradient; the salt which diffuses does not bind to the surface; thegradient creating factor (gradient electrostatic repulsion betweennanoparticles) takes place in the solution on the nanoparticles and noton the surface.

2. Two-Dimensional Diffusion

With the one-dimensional method of diffusion gradient surfaces areacquired in one dimension, e.g. high density of bound particles at oneen of the gradient and a lower density in the opposite end. In anembodiment according to FIG. 6, making of circular gradients ofparticles on a surface is described. FIG. 6A is a side view and FIG. 6Bis a top view.

On the bottom in a Petri dish 601, a plane surface 203 is placed. A saltfree, or almost salt free suspension 602 comprising nanoparticles and amatrix that allows diffusion of nanoparticles but at the same timeprevent convection currents, is poured into the Petri dish. Such asuspension can consist of polysaccharides in a water containing particleform, e.g. gel particles used for gel filtration e.g. Sephadex g-25 orsimilar material. After a free solvent, e.g. water, is removed from theplane suspension layer, a reservoir 603, such as a round piece ofblotter, is placed on the suspension. The reservoir 603 is previouslyfilled with the salt solution 403 with high molarity, for example bysoaking a blotter in such a solution. This system is then given time fordiffusion. The solution 403 will diffuse in the suspension 602 and thecircular diffusion front will after a while reaches the surface 203which eventually will result in a circular surface with a radialconcentration gradient of ions. Finally, the suspension 602 is flushedaway with a solvent, e.g. water. The end result is a circular surface ofadsorbed particles which density is highest in the middle of the surfaceand lowest towards the periphery.

An important aspect of the described invention is the analyticaldynamics, which is the range between that part of the surface gradientwith the highest number of adsorbed particles per surface unit and thepart of the same gradient surface with the lowest number of adsorbedparticles per surface unit. The greater this range is, the moreanalytical information is obtained in adhesion and adsorptionexperiments. A methodological source of error can be electricallyparticles in low concentration in a salt free solvent such as water hasa tendency to bind irregularly to surfaces in unpredictable patterns.Such irregular patterns make the interpretation of additional adhesionand adsorption experiments more difficult. In an embodiment according toFIG. 6C a method to prevent the formation of irregular patterns isprovided. The method comprises mixing electrically charged, surfacebinding particles 200 with the electrically charged particles 604 whichdo not bind to the surface 203. The function of the latter particles isto improve the dispersion of the charged and the binding particles inthe solution in order for pattern of binding not to become irregular.

Typical Experiments and Evaluation Methods

A gradient area with bound particles is normally between 1-50 mm, suchas 1-10 mm. On this surface adsorption experiments with biopolymers andadhesion experiments with cells can be performed. The result of theexperiments can then be correlated to a continuous gradient of boundnanoparticles per surface unit. In simple biopolymer adsorptionexperiments it is possible to use surface sensitive, optical methods. Inexperiments involving whole cells light microscopy can be used fordetailed studies of the cells as well as fluorescent microscopy forstudies of fine details.

In one application of the invention, FIG. 7, the particle gradient isapplied to a surface 700 to which a scale has been added to facilitatethe analysis through a microscope. The scale marks can be vertical 702,horizontal 703 or radial 704. The scales and the scale marks can be indifferent ranges to accommodate different types of analysis e.g. mmrange for ocular analysis or 10-500 micrometers range for the analysisfor light microscopy or SEM which is described in FIG. 7B, 705. Thescale marks can consist of engravings, completely or partly through theparticle-binding surface, e.g. a dithiol modified gold surface, so thatthe underlying substrate, e.g. glass or silica, is exposed.Alternatively, the scale marks can be ridges, e.g. in gold, on aparticle-binding surface, e.g. a thiol silane modified glass or silicasurface. The surface pattern is preferably made with photolithographical techniques.

In another application of the invention, FIG. 8, a nanoparticle gradientsurface 203 or 700 is present on a chip surface 800, e.g. a glass slide,together with two additional, separate surfaces 801 and 802. FIG. 8A isa top view and FIG. 8B and 8C are side views according to twoembodiments. The two additional, separate surfaces are chemicallymodified in such a way that one of the surfaces 802 is given the samesurface chemistry as the surface with the nanoparticles, whereas theother surface 801 is given the same chemistry as the surface thatsurrounds the particles in the gradient. When all three surfaces arepresent in a biological experiment, e.g. bacterial adsorption, theoperator can examine through a microscope whether the bacteria adsorbsto an area of the gradient surface. The operator also obtainsinformation regarding how the bacteria adsorb to surfaces that do notcontain nanoparticles but only unmixed surface chemistries. In this waythe operator can determine whether the adhesion of the bacteria isrelated to the presence of nanoparticles or not, and which particledensity is necessary for adsorption. The different surfaces 203/700,801, an 802 can be separated on the chip 800 with the help of barriers803 in order to facilitate the use and fabrication of the surface. Thebarriers can be made of engravings or spaces between the surfaces203/700, 801, and 802 on the chip 800 in such a way that the underlyingsubstrate 804 is exposed. Alternatively, the barriers can consist ofridges between the surfaces 203/700, 801, and 802, specifically in thosecases where the particle gradient has been applied on the underlyingsubstrate 804.

An application of the invention is a product, e.g. a chip comprising asurface, e.g. a glass slide with the three surfaces mentioned above; 1,gold nanoparticle gradient surface where the gold nanoparticle gradientis manufactured on a dithiol modified gold surface on which freedithiols between the particles has reacted with malemide-PEG and thesurface of the particles has reacted with a functional thiol, e.g.methyl terminated, amino terminated, acid terminated, or peptideterminated; 2, a gold surface which has been modified dithiol andmalemide-PEG; 3, a gold surface which has been modified with the samefunctional thiol as the surface on the particles.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a dithiol modified gold surface on which free dithiolsbetween the particles has reacted with a malemide-conjugated molecule,e.g. methyl terminated, amino terminated, acid terminated, or peptideterminated, phosphorylated, heterocyclics, aromatics, carbonyles,sugars, inorganics, metal containing particles and the surface of theparticles has reacted with a PEG-conjugated thiol 2, a gold surfacewhich has been modified with dithiol and malemide-conjugated molecules,e.g. methyl terminated, amino terminated, acid terminated, or peptideterminated; 3, a gold surface which has been modified with the samefunctional thiol as the surface on the particles.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a dithiol modified gold surface on which free dithiolsbetween the particles has reacted with malemide-PEG; 2, a gold surfacewhich has been modified dithiol and malemide-PEG; 3, a pure goldsurface. With this product, the operator can choose which thiol reagentthat should be used. There is a large number of commercially availablethiols that can affect adhesion, e.g. thiols conjugated with aminogroups, mono- and polysaccharides.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a dithiol modified gold surface; 2, a gold surface whichhas been modified dithiol; 3 a pure gold surface. With this product theoperator can choose both which malemide reagent to be bound between theparticles and which thiol reagent to be bound on the particles. Thepossibilities to alter an experiment will therefore increase further.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a thiol silane modified glass- or silica surface, wherefree thiol silanes between the particles has reacted with malemide PEG,and where the surface on the particles has reacted with the functionalthiol, e.g. methyl terminated, amino terminated, acid terminated, orpeptide terminated; 2, a glass or silica surface which has been modifiedwith thiol silane and malemide-PEG; 3, a gold surface which has beenmodified the same functional thiol as the surface of the particles.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a thiol silane modified glass- or silica surface, wherefree thiol silanes, between the particles, have reacted with malemidePEG; 2, glass- or silica surface which has been modified with thiolsilane and malemide PEG; 3, an unmodified gold surface. With thisproduct the operator can choose which thiol reagent to be used.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a thiol- or aminosilane modified glass- or silicasurface, where the silanes under and between the particles has beenremoved in such a way, e.g. trough plasma treatment, that the particlesare sintered on the glass or silica surface where the surfaces betweenthe particles has reacted with PEG-Silane and the surface of theparticles has reacted with a functional thiol, e.g. methyl terminated,amino terminated, acid terminated or peptide terminated; 2, a glass orsilica surface which has been modified PEG -Silane; 3, a gold surfacewhich has been modified with the same functional thiol as the surface ofthe particles. An application of the invention is a product comprising asurface, e.g. glass slide with the three surfaces mentioned above; 1,gold nanoparticle gradient surface where the gold nanoparticle gradientis manufactured on a thiol- or aminosilane modified glass- or silicasurface and the surface of the particles has reacted with a functionalthiol, e.g. PEG terminated, methyl terminated, amino terminated, acidterminated or peptide terminated; 2, a glass or silica surface which hasbeen modified with thiol- or aminosilane; 3, a gold surface which hasbeen modified with the same functional thiol as the surface of theparticles.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a thiol- or aminosilane modified glass- or silicasurface, where the silanes under and between the particles has beenremoved in such a way, e.g. trough plasma treatment, that the particlesare sintered on the glass or silica surface where the surfaces betweenthe particles have reacted with PEG-Silane; 2, a glass or silica surfacewhich has been modified PEG-Silane; 3, an unmodified gold surface. Withthis product an operator can choose which thiol reagent to be used.

An application of the invention is a product comprising a surface, e.g.glass slide with the three surfaces mentioned above; 1, goldnanoparticle gradient surface where the gold nanoparticle gradient ismanufactured on a thiol- or aminosilane modified glass- or silicasurface, where the silanes under and between the particles has beenremoved in such a way, e.g. trough plasma treatment, that the particlesare sintered on the glass or silica surface; 2, a glass or silicasurface; 3, an unmodified gold surface. With this product the operatorcan choose which surface chemistry and method of modification for thedifferent surfaces.

For the above-mentioned applications the three typical surfaces can inmanufacturing be made separate, and then be combined on the glass slideswith an adhesive. It is also possible to prepare these surfaces directlyon a glass slide.

For simple biopolymer adsorption experiments, surface sensitive opticalmethods such as ellipsometry and surface plasmon resonance (SPR) can beused. A special case of SPR is that so called imaging SPR (iSPR) methodwhich allows for simultaneous quantification of both adsorbingnanoparticles and the following bio adhesion in a complete gradient area(see example). A set up for iSPR analysis of a gradient surface isillustrated in FIG. 9A. An apparatus 900 for imaging SPR such asdescribed in [17], normally controlled with a computer 905, is appliedin contact with an SPR-substrate usually comprising a glass surface 901on which a thin gold layer 902 has been applied. Above the gold layer achamber 904 containing a solution is placed, e.g. a buffer in such a waythat the gold layer is in contact with the solution. The chamber 904 canhave an inlet 907 and an outlet 908 and function as a perfusion systemto transport solution and analytes to and from the surface.

In an application of the invention the gold surface 902 has beenchemically modified with the layer 903 to bind nanoparticles fromsolution, and a gradient of nanoparticles has been applied to thesurface. At analysis the SPR-response from different positions on thegradient surface can be correlated to partial density at this position.In the case bio adhesion, e.g. protein-, thrombocyte-, or bacterialadsorption takes place to the gradient surface this can also be detectedas an additive response, FIG. 9B.

Lately, electrochemical technologies, particularly measurements ofimpedance have been used to study cell interactions with surfaces [18].Electrochemical technologies can also be used to estimate the number ofnanoparticles on an electrode surface. This specifically is true forconducting nanoparticles for instance gold [19]. In an application ofthe invention described in FIG. 10 a nanoparticle gradient is applied ona surface 1000 which is made up of n particle binding surfaces 1001 ofan electrically conducting material which has been modified with thechemistry 1003 to bind nanoparticles. The surfaces 1001 is place on anon conducting substrate 1002 in such a way that the surfaces 1001 anact as electrodes electrically isolated from one and other. Thenanoparticle gradient is manufactured in such a way that the particlebinding electrode surface 1 on the surface 1000 gets a high particledensity while the particle binding electrode surface n on the surface1000 gets low particle density. This is possible by using the surface1000 such as surface 203 in FIG. 4 and let position 0 on the surface1000 coincide x=0 in FIG. 4.

The surface 1000 is applied in contact with an electrolyte, e.g. abuffer, in an electrochemical cell 1004 which also can have an inlet1005 and an outlet 1006 in order to facilitate transportation ofelectrolyte and analyte to the surface 1000. In addition to theelectrodes 1-n localized on the surface 1000, it is necessary for someapplications to add an additional reference electrode 1007 and a counterelectrode 1008 applied in the electrolyte. In some applications theelectrodes 1007 and or 1008 can also be placed on the surface 1000. Allelectrodes 1-n on the surface 1000 and in some cases 1007 and 1008, areconnected individually by a system for electrochemical reference 1009.The system 1009 can be a system capable of different types ofelectrochemical reference, e.g. voltammetry, amperometry, coulometry,impedance spectroscopy or impedance determination. Alternatively thesystem 1009 could be a system designed for a single type ofelectrochemical measurements such as impedance measurements. Theelectrochemical response from the different electrodes on the surface1000 can be measured either between different electrodes on the surface1000, or by using the electrodes 1007 and 1008 in a conventional trielectrode setup [20]. When measuring, the electrochemical response fromdifferent electrodes with different positions on the surface 1000 can becorrelated to the particle density at this position. If bio adhesion,e.g. cell adhesion, takes place to the gradient surface this can also bedetected as an additive, usually a negative, change of theelectrochemical response. If a redox active substance comes in contactwith the surface this can also be detected as an additive, usually apositive change of the electrochemical response.

EXAMPLE 1 Evaluation of Gradual Particle Adsorption with SEM

Gold surfaces with size 11×20 mm were manufactured by evaporation offirst 5 nm Cr and then 200 nm Au on a substrate of SiO2. These werewashed and provided with a monolayer of dithiol according to theprocedure described in detail [1, 8]. In short, the clean gold surfaceswere incubated in a solution of octane dithiol in ethanol where theywere reactivated with dithiolthreitol (DDT). An electrostaticallystabilized gold particle solution with gold particles around 10 nm indiameter was manufactured according to the procedure described in detail[1, 8]. The gold solution was centrifuged at 16000 g to reduce the ionicstrength in the solution, and in order to increase the concentration ofthe particles. After centrifugation the gold particle pellet was dilutedto an approximate particle concentration of 55 nM in pure water with theconductivity of 18.2 MΩ*cm. This particle solution was transferred to acontainer designed gradient manufacturing according to FIG. 5 afterwhich a number of the dithiol prepared surfaces were placed in the samecontainer with a specific distance to the bottom the same. There after acitrate buffer with the concentration 1M and pH 4.0 was placed at thebottom of the gradient container so that the space underneath thesurfaces was filled with this buffer. The citrate buffer was thenallowed to diffuse over the surfaces during 30 minutes after which theprocedure was stopped by emptying the solution from the gradientcontainer from below. In a different application the citrate buffer withthe concentration 50 mM was applied underneath the surfaces and was theallowed to diffuse less than 90 minutes which results in a longergradient with less slope compared to the one obtained with 1 M bufferunder 30 minutes. The surfaces were analyzed with SEM at differentpositions on the gradient surface. A selection of pictures is presentedin FIG. 11.

EXAMPLE 2 Evaluation of Gradual Particle Adsorption by iSPR

Linear gradients with 10 nm gold nanoparticles were prepared asdescribed in example 1 by dithiol chemistry. As a substrate glasssurfaces on which a thin layer of Au, app. 50 nm, had been evaporatedwas used. These surfaces are suitable for analysis by surface Plasmonresonance, SPR, after the manufacture of gradients the surfaces wereplaced in an instrument for imaging SPR which is described in detail in[17]. Two different gradients were analyzed, see FIG. 12. One gradienthad been prepared with 50 mM citrate buffer which has been allowed todiffuse for 90 minutes (“long” gradient), and one gradient had beenprepared with 1 M citrate buffer for 30 minutes (“short” gradient). Eachparticle gradient also had reacted with maleimide-PEG in order minimizebioadhesion between the distributed particles, and with octane thiolabove the particles which makes the surfaces on the particleshydrophobic in order to promote bio adhesion. On each gradient surfacean area of app. 1×5 mm was analyzed. In these areas all essential partsof the gradient was pictured. The SPR wave length presented in the 3Dgraph z-axis is proportional to the surface coverage of goldnanoparticles. In FIG. 13 a line scan of a “short” gradient is presentedtogether with a line scan for a positive control surface, (in this casea surface modified with only octanedithiol), and a negative controlsurface modified with octane dithiol and maleimide-PEG. Each line scanrepresents an average of all scans of the surface.

EXAMPLE 3 Evaluation of Fibrinogen Adsorption and Thrombocyte Adsorptionto Hydrophobic Nanoparticle Gradients with iSPR and FluorescenceMicroscopy

“Short” gradients with 10 nm gold nanoparticles were manufactured ongold surfaces designed for SPR analysis, and were then modified bymalemide-PEG and octanethiol according to example 2 above. This givesgradients of hydrophobic particles against the background of proteinrejecting PEG. The surfaces were analyzed with iSPR. In a sequencefibrinogen (0.5 mg/ml in PBS) for 5 minutes and then thrombocytes(essentially a serum free preparation from a healthy donor) for 30minutes were adsorbed to surfaces with gradients, positive controlsurfaces (only dithiol) , and negative control surfaces (dithiolmodified with malemide-PEG). In FIG. 14 the response from fibrinogen andthrombocyte adsorption is presented for a gradient surface and apositive controlled surface. The blue curve shows the adsorption offibrinogen, the green curve the accumulated response from bothfibrinogen and thrombocytes. The response from the underlying surfaces,corresponding to what is shown in FIG. 13, has been subtracted from theresults in FIG. 14. Note that the positive surface adsorbs bothfibrinogen and thrombocytes homogeneously across the surface, while thegradient surface adsorbs both protein and thrombocytes gradually. Thenegative control surfaces gave no significant response. Afterthrombocyte adsorption the surfaces were washed with PBS buffer andfixated for 15 minutes with 2% glutaraldehyde. The surfaces were stained(staining of the actin skeleton) according to a normal protocol and wereanalyzed fluorescence microscopy at different positions at the surfaces.FIG. 15 shows representative thrombocytes at positions with high (A) andlow (B) particle coverage respectively.

EXAMPLE 4 Evaluation of Microbial Adhesion to Hydrophobic NanoparticleGradients.

“Long” gradients with 10 nm gold nanoparticles were manufactured on goldsurfaces modified with malemide-PEG and octane thiol according toexample 2 above. This gives gradients with hydrophobic particles againstthe background of protein rejecting PEG. Fimbriated E. coli wereadsorbed to the surfaces under static conditions, and were exposed to acontrolled wash for 10 minutes. Remaining bacteria were stained withacridine orange and DAPI after which the surfaces were analyzed undermagnifying glass and fluorescence microscopy. FIG. 16 shows a section ofa gradients surface with adsorbed E. coli stained with acridine orangeat low magnification together with a positive control surface (octanethiol) and negative control surface (dithiol modified withmalemide-PEG). The surface coverage of nanoparticles at differentpositions in the gradient was determined by SEM. The relative surfacecoverage is shown in each picture. The inserted picture shows twobacteria stained with DAP at greater magnification. The distribution ofbacteria changes dramatically at 20% surface coverage.

Although the present invention has been described above with referenceto specific embodiments, it is not intended to be limited to thespecific form set forth herein. Rather, the invention is limited only bythe accompanying claims and, other embodiments than the specific aboveare equally possible within the scope of these appended claims.

In the claims, the term “comprises/comprising” does not exclude thepresence of other elements or steps. Furthermore, although individuallylisted, a plurality of means, elements or method steps may beimplemented by e.g. a single unit or processor. Additionally, althoughindividual features may be included in different claims, these maypossibly advantageously be combined, and the inclusion in differentclaims does not imply that a combination of features is not feasibleand/or advantageous. In addition, singular references do not exclude aplurality. The terms “a”, “an”, “first”, “second” etc do not preclude aplurality. Reference signs in the claims are provided merely as aclarifying example and shall not be construed as limiting the scope ofthe claims in any way.

References

1. Lundgren A. O., et al., Self-Arrangement Among Charge-Stabilized GoldNanoparticles on a Dithiothreitol Reactivated Octanedithiol Monolayer.Nano Letters, 2008. 8(11): p. 3989-3992.

2. Adamczyk Z., et al., Structure and ordering in localized adsorptionof particles. Journal of Colloid and Interface Science, 1990. 140(1).

3. Hanarp P., et al., Control of nanoparticle film structure forcolloidal lithography. Colloids and Surfaces A: Physicochemical andEngineering aspects, 2003. 214: p. 23-36.

4. Johnson C. A. and Lenhoff A. M., Adsorption of charged latexparticles on Mica studied by atomic force microscopy. Journal of Colloidand Interface Science, 1996. 179: p. 587-599.

5. Semmler M., et al., Diffusional deposition of charged latex particleson water-solid interfaces at low ionic strength. Langmuir, 1998. 14: p.5127-5132.

6. Kooij E. Stefan, et al., Ionic strength mediated self-organisation ofgold nanocrystals: an AFM study. Langmuir, 2002. 18: p. 7677-7682.

7. Verwey E. J. W. and Overbeek J. Th. G., Theory of the stability oflyophobic colloids. 1948, Amsterdam: Elsevier Publishing Company Inc.

8. Lundgren A. O., PCT/SE2009/051060. 2009.

9. Arnold M., et al., Activation of integrin function by nanopatternedadhesive interfaces. ChemPhysChem, 2004. 5: p. 383-388.

10. Michel R., et al., A novel approach to produce biologically relevantchemical patterns at the nanometer scale: selective molecular assemblypatterning combined with colloidal lithography. Langmuir, 2002. 18: p.8580-8586.

11. Elwing H., et al., A wettability gradient-method for studies ofmacromolecular interactions at the liquid solid interface. Journal ofColloid and Interface Science, 1987. 119(1): p. 203-210.

12. Kim M. S., Khang G., and Lee H. B., Gradient polymer surfaces forbiomedical applications. Progress in polymer science, 2008. 33(1): p.138-164.

13. Morgenthaler S., Zink C., and Spencer N.D., Surface-chemicaland—morphological gradients. SOFT MATTER, 2008. 4(3): p. 419-434.

14. Liedberg B. and Tengvall P., Molecular gradients ofomega-substituted alkanethiols on gold—preparation and chracterization.Langmuir, 1995. 11(10): p. 3821-3827.

15. Grabar Katherine C., et al., Kinetic control of interparticlespacing in Au colloid-based surfaces: Rational nanometer-ScaleArchitecture. Journal of the American Chemical Society, 1996. 118: p.1148-1153.

16. Arnold M., et al., Induction of cell polarization and migration by agradient of nanoscale variations in adhesive ligand spacing. NanoLetters, 2008. 8(7): p. 2063-2069.

17. Andersson O., et al., Gradient Hydrogel Matrix for Microarray andBiosensor Applications: An Imaging SPR Study. Biomacromolecules, 2009.10: p. 142-148.

18. K→owino I. O. and Sadik O. A., Impedance spectroscopy: A powerfultool for rapid biomolecular screening and cell culture monitoring.Electroanalysis, 2005. 17(23): p. 2101-2113.

19. Zhao J. J., et al., Nanoparticle-mediated electron transfer acrossultrathin self-assembled films. Journal of Physical Chemistry B, 2005.109(48): p. 22985-22994.

20. Bard A. J. and Faulkner L. R., Electrochemical Methods. 2:nd ed.2001: John Wiley & Sons Inc.

1. A method for preparing a continuous gradient of deposited and electrically charged nanoparticles along a solid surface, wherein the number of deposited and electrically charged nanoparticles per unit area of the surface is high on one end of the surface and low on the opposite end of the surface, and wherein the distance between the deposited particles, at the time of deposition, is regulated through electrostatic repulsion between the nanoparticles in a solution, characterized in that the degree of electrostatic repulsion of particles in the solution is obtained by a diffusion of a salt solution into the solution comprising nanoparticles.
 2. The method of claim 1, wherein the diffusion of salt solution is obtained by forming a layer of a salt solution with relatively high density and concentration under a layer of substantially salt free solution comprising nanoparticles, and that the continuous gradient is regulated by the time of diffusion and concentration of salt in the salt solution.
 3. The method according to claim 1, wherein the diffusion of the salt solution is obtained by keeping the salt solution in a reservoir, placed into contact with the suspension of nanoparticles, said suspension further comprising a matrix allowing diffusion of nanoparticles but prohibiting convection currents, and which suspension of nanoparticles is in contact with the solid surface.
 4. The method according to any claim 1, wherein the nanoparticles consist of metal, ceramics, glass, or polymer material, and wherein the solid surface consists of metal, ceramics, glass, or polymer material.
 5. (canceled)
 6. (canceled)
 7. The method according to claim 1, wherein the surface is gold, with bound dithiol reagent and the nanoparticles are covalently bound to thiol groups of the dithiol molecule bound to the gold surface.
 8. The method according to claim 1, wherein negatively charged but non-surface binding particles are mixed with surface binding nanoparticles.
 9. The method according to claim 1, further comprising adding a first separate surface and a second separate surface to the surface, wherein the first separate surface has a surface chemistry similar to the nanoparticle and the second separate surface has a surface chemistry similar to the surface.
 10. The method according to claim 1, further comprising adding scale marks to the surface.
 11. A surface with a continuous gradient of deposited and electrically charged nanoparticles.
 12. The surface according to claim 11, wherein the gradient length is between 1 mm and 50 mm.
 13. The surface according to claim 11, wherein the nanoparticles have an average diameter between 10 and 60 nm.
 14. The surface according to claim 11, wherein the gradient is linear.
 15. The surface according to claim 11, wherein the average center-to-center distance of the nanoparticles is about 10-60 nm in one end of the gradient and about 100-150 nm in the other end of the gradient.
 16. The surface according to claim 11, wherein the nanoparticles and/or the surface consists of metal, ceramics, glass, or polymer material.
 17. The surface according to claim 11, wherein the nanoparticles and/or the surface have a compound conjugated to them.
 18. The surface according to claim 17, wherein the compound is selected from the group consisting of dithiol groups, thiol groups, PEG, and aminosilane.
 19. A device for analysis of adhesion phenomena, comprising a gradient surface, according to claim 11, a first separate surface and a second separate surface, wherein the surfaces are separated and the first separate surface has a surface chemistry similar to the nanoparticle and the second separate surface has a surface chemistry similar to the gradient surface.
 20. The device according to claim 19, wherein the nanoparticles, the gradient surface, the first separate surface or the second separate surface have the same or different compound/s conjugated to them, wherein the compound/s is/are selected from the group consisting of thiol groups, PEG, and aminosilane.
 21. Use of the device according to claim 19, for adhesion analysis.
 22. Use according to claim 21, wherein the analysis is based on Surface Plasmon Resonance (SPR), electrochemistry, light microscopy or scanning electron microscopy (SEM). 